It is our hypothesis that Phospholipase A2 (PLA2) activity adversely affects cellular function after brain ischemia and plays a critical role in the process of brain infarction. One goal of this proposal is To determine the mechanisms of regulation of the 100 kDa cytosolic PLA2 (cPLA2) by excitotoxins and evaluate the role of cPLA2 activation and translocation in excitotoxicity in neuronal cultures. We have demonstrated that glutamate markedly enhances cPLA2 enzymatic activity in neuronal cultures. We will evaluate the role of metabotropic and NMDA receptor activation and resulting intracellular signalling events in cPLA2 activation. In primary neuronal cultures as well as polarized human neurons derived from NTera 2 cells we will alter the cellular expression of cPLA2 in order to evaluate the importance of this enzyme in neuronal injury. The Ca2+ translocation domain of cPLA2 will be mutated and effects of this alteration of Ca2+ mediated arachidonic acid release and vulnerability to excitotoxic injury will be evaluated. A second goal of the proposal is To purify, clone, and express novel neuronal forms of PLA2 and study their functional characteristics, role in ischemic brain injury and effects of overexpression on susceptibility to excitotoxins. We have partially purified a novel large molecular mass Ca2+ independent form of PLA2 from the bovine brain. We will complete this purification, characterize the enzyme, and identify how the enzyme activity is altered by ischemia in vivo and glutamate in vitro. We have also cloned a novel cDNA from the mouse brain which encodes a putative PLA2. Effects of overexpression of these novel forms of PLA2 on neuronal vulnerability to excitotoxicity will be evaluated. Our third goal is To localize expression of PLA2 enzymes in the brain under normal conditions and in experimental ischemia. With the antibody we have recently raised to cPLA2, as well as other antibodies raised to novel brain forms of PLA2, immunocytochemical and immunogold studies will be performed to identify which cell type(s) express the enzyme, where in the cell it is expressed, and how this expression is altered in cerebral ischemia. Our fourth goal is To evaluate whether p42MAP and calcium/calmodulin kinase II associate with cPLA2 intracellularly, phosphorylate cPLA2 and regulate enzymatic activity. We will use a """"""""hybrid trap"""""""" interaction system to determine whether cPLA2 associates with these kinases intracellularly and define the regions of cPLA2 which are important for this association. Defining the PLA2 forms which are important for brain injury and their means of regulation, will permit a directed approach to development of strategies to alter the activity of the enzyme(s) and protect the post- ischemic brain from infarction.
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